Objective Pistil abortion is a key factor limiting Xanthoceras sorbifolium yield. miR167 may participate in the auxin pathway by regulating ARF genes, but its specific function in the pistil abortion of Xanthoceras sorbifolium remains to be elucidated. This study aimed to investigate the regulatory mechanism of the miR167-XsARF-IAA module during pistil abortion in male flowers of X. sorbifolium Bunge, and to clarify the function of the key IAA pathway gene XsARF6 regulated by miR167, so as to lay a preliminary theoretical foundation for dissecting the regulatory mechanism of pistil abortion in this species.

Method Pistils of female and male flowers at three key developmental stages were sampled for whole-transcriptome sequencing. The key xso-miR167 was identified by integrated miRNA and mRNA analyses. Biological validation and hormone measurement were performed to analyze the expression patterns of miRNAs and mRNAs were analyzed. IAA content in kernels of X. sorbifolium overexpressing xso-miR167 and phenotypes of transgenic Arabidopsis thaliana were determined to elucidate the dynamic regulation of the miR167-XsARF-IAA module.

Result (1) A total of 481 miRNAs and 2 266 predicted target genes were identified across the three key stages of pistil abortion, with differentially expressed miRNAs showing clear stage-specific patterns, indicating that different miRNAs play critical regulatory roles during the abortion process. (2) The expression of xso-miR167 increased continuously during pistil development in male flowers, significantly repressing its target gene XsARF6.2, and their expression trends were negatively correlated, suggesting that this regulatory pair is a key factor in pistil abortion. (3) xso-miR167 overexpression significantly decreased kernel IAA content compared with wild-type and empty-vector controls. (4) In transgenic A. thaliana, pollen viability decreased from 90.9% in the wild type to 43.68% and 62.25%, stigma diameter and lengths of pistils and stamens were significantly reduced, flowering was delayed, and floral organ development was abnormal.

Conclusion The miR167-XsARF-IAA regulatory module exists during pistil development in male flowers of X. sorbifolium. miR167 participates in regulating the biosynthesis, metabolism or transport of IAA by inhibiting the expression of XsARF6.2, and plays a key role in pistil abortion of X. sorbifolium. This study provides a new perspective on the molecular mechanism of auxin-regulated pistil development in X. sorbifolium and offers a theoretical basis for fundamental research and breeding applications.